PROJECT SUMMARY/ABSTRACT ? No change from original proposal Many prokaryotes use extracellular appendages, notably the flagella and pili, for motility and colonization of biotic and abiotic substrates. Both flagella and pili are well-known virulence factors for numerous pathogenic bacterial species. Here we aim to determine two things: 1) how redundancy of the flagellins in multi-component flagellar filaments alters overall flagellum structure and function, and 2) the structural variation that exists between several classes of type IV pili at the macromolecular level. To answer these questions, we are using several bacterial species, Vibrio cholerae and Vibrio vulnificus, human pathogens that cause severe gastroenteritis, diarrhea, and death; and Caulobacter crescentus, a well-established non-pathogenic model system. We are using these organisms to build upon our previous structural studies of the polar appendages of C. crescentus and to develop Vibrio species as model systems for structural studies of pathogens. Together, these studies will provide new information regarding the structural and functional diversity of prokaryotic flagella and pili. The three areas to be investigated in this project are: 1. Determine the structural and functional implications of multi-flagellin flagella in pathogenic and nonpathogenic bacteria. Experiments will determine the structure of flagella from wild type and mutant C. crescentus, V. cholerae, and V. vulnificus in situ through cryo-ET of frozen hydrated cells. Cryo-EM and helical reconstruction approaches will define the high-resolution structure of flagella isolated from C. crescentus, V. cholerae, and V. vulnificus. 2. Determine the structures of several classes of type IV pili in intact bacterial cells. Experiments in this aim will determine the in situ structure of the T4bP of V. cholerae and V. vulnificus and Flp-type pili of C. crescentus through cryo-ET and sub-tomogram averaging. We will characterize structural changes resulting from mutants to the pilus filament or pilus complex and their impact on pilus structure, pilin secretion and possible retraction, and complex integration into the cell membrane. 3. Continue the development of cryo-electron tomography technology to generate improved structures of prokaryotes and their appendages. We aim to optimize our newly installed Zernike phase contrast electron microscope for cryo-ET applications. Our goal is to develop ZPC cryo-ET by optimizing the thin films used for Zernike phase plates and developing full automation of biological data collection. In addition, this state of the art technology calls for the development of new software and improvement of already existing algorithms for a more efficient and biologically relevant filtering and `denoising' of cryo-ET data.